ABSTRACT Built on the success of collaboration of our multi-disciplinary team of investigators from UCLA Medical center, Jonsson Comprehensive Cancer Center (JCCC), and California Nanosystem Institute (CNSI), we have developed a potentially new cancer diagnostic platform, called ?Mechano- or Nanocytology?. The basic hypothesis is that as the fundamental lethal features of cancer are tumor cell invasion and metastasis, directly measuring the mechanical properties of cells associated with tumor invasion and metastasis may provide an accurate diagnostic and prognostic marker for cancer. Cancer cell mechanical signature including cell stiffness, elasticity, deformability and permeability, among others, can be quantitatively measured in label free manner at the single cell level (S. Cross, Nature Nano, 2007; Gossett, PNAS, 2012; Tsai, Science Trans. Med., 2013; Rowat, J Biol Chem 2013; Qi, Sci Rep 2015), using state of the art technologies such as Atomic Force Microscope (AFM), Deformability Cytometry (DC), and Parallel Microfiltration (PMF). These methods collectively enable robust measurements which can be implemented in a clinical setting. While data from us and others demonstrated metastatic cancer cells show rather distinctive mechanical features to morphologically similar but normal cells, there have been limited studies about how the mechanical changes occur during the multi-step carcinogenic process, or specifically focusing on a cancer type to explore the possibility of using these markers to address specific clinical questions. These important questions are essential for the long-term success of the nanocytology program. In this R21 application, we will study the changes in the mechanotypic profile of cells at various stages of cancer development and progression using a unique in vitro multi-step bladder carcinogenic model (Aim 1), and test the performance of the mechanotypic marker specifically in cytological diagnosis of urothelial carcinoma in bladder irrigation cytological fluid samples (Aim 2). For Aim 2, we will also determine if the mechnotypic markers can be used to distinguishing pre-invasive carcinoma in situ (CIS) from invasive urothelial carcinoma, a question that has substantial clinical implication in determining whether patient should receive cystectomy of not. The study will provide essential preliminary data for our eventual goal of developing the mechanotypic signature as a novel adjunct biomarker for cancer early detection. It also may lead to the development of an entirely new technology platform for quantitative, robust, and accurate determination of cancer cell behavior at the single cell level.